Recombinant adenoviral vectors and their utility in the treatment of various types of fibrosis: hepatic, renal, pulmonary, as well as hypertrophic scars

ABSTRACT

The invention encompasses the use of gene therapy for the treatment of different kinds of fibrosis in human beings. Specifically, the invention encompasses the use of therapeutic genes specifically directed to target organs to revert and/or prevent the development of the fibrosis process. The invention further encompasses genes encoding for proteins including human MMP-8 active and latent, MMP-1, MMP-2, MMP-9 and MMP-13; human uPA wild type and/or modified (or its truncated version), the truncated receptor for TGF-β type II and Smad-7, which can be directed by adenovirus and/or other recombinant vectors that cannot transduce (i.e., infect) others organs. The gene therapy of the invention further encompasses treating disorders including renal fibrosis, pulmonary fibrosis, hypertrophic and keloid scars (i.e., skin fibrosis), and other kinds of fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/098,359, filed Mar. 18, 2002, now abandoned, which is a continuationof the national stage designation of PCT/MX00/00035, filed Sep. 14,2000, the disclosures of which are incorporated herein in theirentirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the creation of RECOMBINANT ADENOVIRALvectors bearing exogenous genes that encode for therapeutic proteinsuseful in the treatment of HEPATIC cirrhosis and generalized FIBROSIS,such as renal FIBROSIS, pulmonary FIBROSIS, HYPERTROPHIC scars andkeloid of the skin, and/or in other target organs susceptible to sufferfrom it. It also relates to a mechanism of tissue-specific recognitionof the affected cells by means of delivery of therapeutic genes tocirrhotic organs.

Moreover, the invention provides an effective way for the treatment offibrosis through the employment of recombinant adenoviral vectors whichare claimed here, as well as the process to prepare these vectors, thepharmaceutical composition that contains them, and their therapeuticuses in the treatment of several fibrosis, which has great commercialexpectancy in the pharmaceutical industry and also presents an importantalternative as gene therapy for the treatment of chronic-degenerativediseases characterized by fibrosis, with great therapeutic applicationin the field of Medicine.

INTRODUCTION Physiopathology of Hepatic Cirrhosis

Hepatic cirrhosis is a disease resulting from hepatic chronic damage.Damage might be toxic (chronic ingestion of alcohol), infectious (viralhepatitis, mainly by hepatitis B and/or C virus), immunological,(primary biliary cirrhosis), by biliary obstruction, (secondary biliarycirrhosis), metabolic (Wilson's disease). All forms of cirrhosis havecharacteristics in common: synthesis and excessive deposition ofproteins of extracellular matrix (ECM), (mainly collagen I and to alesser extent collagens IV and III), and consequently the formation ofnodules of hepatocytes, abnormal vascularization and portal hypertension(Antoni P P, Ishak K G, Nayak N C, Poulsen H E, Scheuer P J, Sobin L H.The morphology of cirrhosis: definition, nomenclature, andclassification. Bulletin of the World Health Organization. 1977;55:521-540 y Scott L. Friedman The cellular basis of hepatic fibrosis:Mechanisms and treatment strategies. The New England Journal of Medicine1993, vol. 328 No. 25:1828-1835). These physiopathological processeslead to an alteration in the blood supply and in consequence in thenutrition of hepatic cells. Regardless of the ethiological agent andmorphologic differences, all forms of cirrhosis have as a common end,hepatic failure causing the patient's death.

As a consequence of the excessive deposition of collagen proteins in thesub-endothelial space of the sinusoids (Space of Disse), various changesoccur in the hepatic microenvironment: loss of hepatocyte villi,formation of a basement membrane composed by collagens IV and I coveringthe sinusoids, and loss of the fenestration of endothelial cells whichforms the sinusoids. All this process is known as “capillarization” ofthe sinusoids. (Scott L. Friedman The cellular basis of hepaticfibrosis: Mechanisms and treatment strategies. The New England Journalof Medicine 1993, vol. 328 No. 25:1828-1835). Thus, the liver is notable to maintain the physiologic concentration of solutes in theterminal hepatic vein, in other words, HEPATIC failure sets in. Thiscapillarization, with the formation of the continuous endothelia(collagen of basement membrane) and the accumulation of other collagenicproteins, represents a barrier to the normal and bi-directional exchangeof molecules between the plasma and hepatocytes, as can be appreciatedin FIG. 1, where hepatic cirrhosis is characterized by the accumulationin the liver of type I collagen. With an excessive deposition of thisprotein, the free exchange of nutrients between blood and liver cells isimpeded, the inactivation of toxic agents by this organ can not becarried out, becoming this the main cause of the pathophysiology of thedisease. To date, no therapeutic agent that could revert and/or preventwith a 100% effectiveness the progressive accumulation of hepaticcollagen has been described.

Such physiopathological alterations presented in hepatic cirrhosis areconstant and common for the organs that also undergo fibrosis, such as,lung, heart, kidney, skin, among others, which should be not consideredas limitations of the scope of protection of this invention. Therefore,the methodology presented here for the treatment of hepatic cirrhosiscould be applied also to those organs that are susceptible to, or areaffected by fibrosis.

Viral Vectors and Hepatic Gene Therapy

This technology can be implemented with viral or non-viral vectors.Previous studies have been designed using plasmids and liposomes(DOTMA), cationic and anionic, etc. Among the methods employing viralvectors, the most commonly used include the use of retrovirus andadenovirus.

In a number of protocols, retroviral vectors have been used to introducegenes in hepatocytes (JT, and Curiel D T, Adenoviruses as Vectors forgene Therapy. Science and Medicine/1997 44-53). However, precautionshave to be taken since these vectors can generate potentialreplication-competent viruses. Among the advantages of these vectors istheir ability to integrate their genome in a stable way in thechromosomes of the guest cell, which confers the possibility ofexpression, in an indefinite way, of the therapeutic transgene cloned inthe retrovirus. On the other hand, up to date, no study has reportedincidences of mutagenesis by insertion or activation of oncogenes by theincorporation of the replication-deficient retrovirus. Nevertheless, theuse of retroviral vectors to transduce genes to the liver is limited forthe following considerations: 1) these vectors infect only cells whichactively divide and 2) very low viral particles titers are obtained inthe packing cell lines used to amplify these viruses (Graham F L, andVan Der Eb A J. A New Technique for the Assay of Infectivity of HumanAdenovirus 5 DNA. Virology 1973, 52:456-467). These two limitations havebeen successfully overcome in other Gene Therapy protocols through theinduction of hepatocytes proliferation “in vivo”, through the useHepatic Growth Factors and through partial hepatectomy, surgicalprocedure by which the removal of 70% of liver mass induces division ofthe remaining hepatic cells “in vivo”. The use of Lentiviral vectors haspermitted to overcome partially said limitations, because they are ableto transduce cells which are not actually dividing.

BACKGROUND OF THE INVENTION

Hepatic cirrhosis is a chronic illness of the liver, where diffuse cellnecrosis and a limited regeneration of parenchymal hepatic cells resultin diffuse percentage increase of connective tissue, causing thedistortion of lobular hepatic architecture and inducing hemodynamicalterations. Therefore, some strategies for the treatment of hepaticcirrhosis could include the prevention and/or reversion of the“fibrogenic process”, stimulation of hepatic mitosis and re-arrangementof the architecture of hepatic tissue. The documents of the state of theart related to the present invention are mentioned hereinafter only asreferences.

U.S. Pat. No. 5,240,846 refers to the use of gene therapy called “CFTR”,which induces a stable correction of the regulation of the chlorinechannel. This defect is present in epithelial cells. In said invention,adenoviral recombinant vectors are used as well as plasmidic vectors.However, it does not have any association with the therapeutics genes ofthe present invention. Likewise, U.S. Pat. No. 5,910,487, describes theuse of plasmidic vectors for sending therapeutic molecules, but there isno association with the delivery of genes of metalloproteases MMP-8latent and/or active, MMP-1, MMP-2, MMP-9, MMP-13; or “uPA (wild typeuPA and/or its modified versions) or “Smad7” or the truncated receptorsfor transforming growth factor-β (TGF-β type II) as presented here. U.S.Pat. No. 5,827,703 refers to the use of adenoviral vector and modifiedadenoviral vector to send genes, however none of these vectors containthe genes used in the present invention for the treatment of fibrosis.

U.S. Pat. No. 5,770,442 claims the use of a recombinant adenovirus thatcontains one gene directing the expression of a protein called “fiber”or a protein called “Fiber-chimera”, however said patent does notspecifically mention, which one is the therapeutic gene. Also, a methodof gene therapy involving the use of such adenovirus and a vector oftransference for the generation of such recombinant adenovirus ispresented. However, nothing is mentioned with regard to the use oftherapeutic genes cloned and inserted in recombinant adenoviral vectorsused in this invention in fibrotic livers, or to other target organssuch as kidney, lung, and hypertrophic scars and others. Thesetherapeutic genes are the gene that codes for human metalloproteasesMMP-8, latent and/or active, MMP-1, MMP-2, MMP-9 and MMP-13; humanurokinase Plasminogen Activator (wild type and/or modified huPA), Smad7,and the truncated receptor for TGF-β type II, claimed herein. Othermembers of the family of genes represented are also included.

U.S. Pat. No. 5,166,320 refers to the use of a targeted delivery systemto introduce exogenous genes in mammalian hepatic cells. But there is noassociation with putative genes directly sent to cirrhotic livers or tofibrotic kidney or lungs.

U.S. Pat. No. 5,872,154, describes a method to reduce the immuneresponse induced by an adenoviral recombinant vector and a selectedimmune modulator, which functions by inhibiting the formation ofneutralizing antibodies and/or reducing the death of the virallyinfected cells.

U.S. Pat. No. 5,871,982, is directed to a hybrid vector, in which aportion of an adenovirus is included, together with a portion of anadeno-associated viral vector that contains a selected transgene. Ahybrid virus consisting of the union of a conjugate with a polycation toa gene mesh of the adeno-associated viral vector to form a simpleparticle is also described. This is contrary to the present invention inwhich no hybrid viruses are employed, only adenoviral vectors. Besides,in the above-mentioned patent the gene, transgene or therapeutic geneused is not stated.

U.S. Pat. No. 5,856,152 is directed to the creation of a hybrid vectorwhich contains the portion of an adenoviral vector in combination withan adeno-associated virus and a selected gene. Thorough it largequantities of recombinant vectors are produced, but they are notcarrying cloned therapeutic genes as is described in this invention, inwhich specific therapeutic genes for the treatment of renal and hepaticfibrosis and hypertrophic scars are used.

U.S. Pat. No. 5,547,932 claims a compound of complexes of nucleic acidsfor transfecting eucaryotic cells. These complexes are formed by nucleicacids and another substance with affinity for nucleic acids andoptionally an internalizing factor, such as a virus or a component ofthe virus that can be conjugated. It also uses components of specificadenoviral vectors or specific viruses such as Ad2 or Ad5, but does notmention the genes that are internalized in the cell cytoplasm andeventually in the nucleus of these eucaryotic cells. Similarly, U.S.Pat. No. 5,521,291, is related to conjugated adenovirus bound through anantibody to a substance with affinity to nucleic acids. In this wayrecombinant genes are transported to the interior of eucaryotic cells.These conjugated complexes and nucleic acids are internalized in thecell, but the genes that can be sent are not specifically mentioned. Insaid patent, contrary to what is described in the instant invention, theuse of such adenovirus to treat fibrosis or hepatic cirrhosis or anyanother type of fibrosis is not mentioned.

U.S. Pat. No. 5,585,362, relates to an improved adenoviral vector andmethods to obtain and use such vectors. The use of adenoviral vectors isnot mentioned in said patent. However the adenoviral vectors describedin the present invention were used like vectors for sending therapeuticgenes.

U.S. Pat. No. 5,756,086, claims an adenovirus, which is represented by aprotein called “fiber”, the adenovirus also includes a ligand, that isspecific for a receptor located in a specific cell type. This adenoviruscan have at least a portion of this protein called “fiber” and it can beremoved and replaced with a ligand, which is specific for a receptor inspecific cells of the economy, such as hepatocytes. This adenovirus caninclude a gene that codes for a therapeutic agent. Based on the previousstatement, the outstanding technical difference of the instant inventioncompared to the state of the art, is the specificity of the therapeuticagent as human metalloproteases MMP-8 active and latent, MMP-1, MMP-2,MMP-9 and MMP-13; human uPA (urokinase Plasminogen Activator, wild typeand/or modified), the truncated receptor for TGF-β type II and “Smad7”for the treatment of various fibrosis.

U.S. Pat. No. 5,895,759 claims a tissue-specific vector (liver) for genetherapy that can be used to send genes to a damaged liver. These vectorsare chemically or enzyme coupled to a promoter and can also be coupledto an antibody packaged in a polypeptidic envelope. Besides, the vectoror the virus to be assayed is the hepatitis B virus. Thus the sending ofgenes to damaged livers described in this patent makes use of a systemcompletely different from the one of this invention, and there is norelation with the process of fibrosis or cirrhosis to be treated.

U.S. Pat. No. 5,559,099 describes an adenoviral recombinant vector thatcontains a chimeric protein from the adenovirus called pentona, whichincludes a non-pentona sequence and a therapeutic gene to develop a genetherapy method involving the use of such adenovirus, transferenceadenoviral vectors for the recombination of such adenoviral vectorscontaining a therapeutic gene.

U.S. Pat. No. 5,885,808 claims also the use of adenovirus with bondingmolecules of adenovirus to different cells, the molecules of which havebeen modified, as in U.S. Pat. Nos. 5,846,782 and 5,712,136, in whichadenoviral vectors are employed, which have been modified to containdifferent peptidic domains.

Finally, U.S. Pat. No. 5,670,488 relates to vectors for gene therapy,which are especially useful for cystic fibrosis and also mentions thedevelopment of methods for the use of these vectors. The possiblerelation of the instant invention to the mentioned state of the artrefers to the use of adenoviral vectors, that can be modified, as wellas the use of inducible promoters driving the expression of genes to beinserted in these adenoviral vectors. However, the technicalcharacteristics of the present invention are focused on the specific useof therapeutic genes to treat fibrosis of different kinds: hepatic,renal and pulmonary fibrosis, as well as hypertrophic scars.

The importance of the present invention, contrary to the state of theart described in the above-mentioned documents, is based on thetechnical characteristics of the invention itself, as well as on theadditional advantages derived from the same, which are described withmore details below.

Adenoviral Vectors

In the instant invention, the use of adenoviral vectors was determinedbased on several considerations: 1) these vectors can be generated tovery high titers of infectious particles per ml.: (10⁹-10¹⁰); 2) theyinfect a great variety of cells, however, when they are administeredi.v., most of them are located in the hepatic organ; 3) they transferefficiently genes to cells that are not dividing, and 4) they are seldomintegrated in the guest genome, which avoids the risk of cellulartransformation by insertional mutagenesis (Douglas J T, and Curiel D T.Adenoviruses as Vectors for gene Therapy. Science and medicine,March/April 1997. 44-53 and Zern A M, and Kresina T F. Hepatic Drugdelivery and Gene Therapy. Hepatology 1997, Vol. 25, No. 2, 484-491).

Adenovirus are probably the most promising vehicles or vectors for thedelivery of genes in the protocols of gene therapy in human beings,since they possess a unique attribute that provides them great stabilitywhen they are administered into the bloodstream. This specificcharacteristic permits them to be efficiently used in clinical trialswith a comfortable i.v. administration for the patient. (Douglas J T,and Curiel D T. Adenoviruses as vectors for Gene Therapy. Science andMedicine, March/April, 1997, 44-53).

Adenoviruses are double stranded DNA viruses. They have an icosahaedricstructure, infect a great variety of mammalian cell types, and supportthe ubiquitous expression of a specific receptor in the cell surface notyet identified. Its union to cells occurs by means of the proteincomponent of the capside and the virus enters into the cell byreceptor-mediated endocytosis.

More than 40 different human serotypes of adenovirus have beenidentified, of which type 2 (Ad2) and 5(Ad5) have been more extensivelystudied and, therefore, more widely used as vectors for gene therapy. Avery important characteristic of these two Ad serotypes is that theyhave never been associated with malignant human processes.

The strategy for the creation of recombinant adenovirus is based on theorganization of the adenoviral genome. The expression of the adenoviralgenes occurs in two phases, early and late, that are defined by the timeof replication of the adenoviral genome. The early genes encodethemselves in 4 distinct transcriptional units: E1, E2 and E4 encode foressential regulatory proteins that induce the replication of theadenoviral DNA. The gene E3 is a non-essential gene. The products of thelate genes include the main proteins of the capside, which aretranscribed from a unique promoter. (Graham F L, and Van Der Eb A J. Anew technique for the assay of infectivity of human adenovirus 5 DNA.Virology 1973, 52:456-467).

The recombinant adenoviruses are generated by introduction of theexogenous gene or sequence of DNA of interest in substitution of theadenoviral genome regions required for the replication of the virus. Theadenoviral recombinant vectors present deletions in E1 and E3 genomeregions. Recombinant adenovirus generation is conducted both through thereplacement of E1 or E3 regions or through the insertion of theexogenous gene between the E4 region and the right extreme of theadenoviral genome. Vectors based on the insertion of the exogenous genat the right extreme of the adenoviral genome or by the replacement ofthe E3 region maintain their replication capability. On the contrary,the substitution of early region E1 produces a faulty vector in itsreplication capability, that, therefore, can spread only in a cell linethat supplies in “trans” the absent functions of the replaced adenoviralregion, or in presence of a collaborator virus. Of these, the mostcommonly used as gene transference vectors are the replication-deficientadenovirus (Douglas J T, and Curiel D T. Adenoviruses as vectors forGene Therapy. Science and Medicine, March/April, 1997, 44-53).

The creation of adenoviral vectors, as well as their application for thetreatment of fibrosis, are shown in the examples described hereinafter.

OBJECTS OF THE INVENTION

Hereinafter, the objects and advantages derived from this invention arepresented.

An object of the present invention is to provide a procedure to preparerecombinant adenoviral vectors pAdGFP-MMP-8, by means of the cloning ofthe reporter genes: lac-7 and GFP and the therapeutic gene ofcollagenase or metalloprotease MMP-8 in its latent and/or active forms.

Another object of the invention is to provide an adenoviral recombinantvector with an exogenous gene or DNA sequence of interest that encodesfor therapeutic proteins useful in the treatment of the generalizedfibrosis, in target organs susceptible to suffer from it. Such genesare, but are not limited to MMP-8 active and latent, MMP-1, MMP-2, MMP-9and MMP-13; and uPA (wild type and/or modified).

Also, in the present invention, pharmaceutical compositions are providedwhich contain the recombinant adenoviral vectors in quantitiestherapeutically effective of viral particles for the treatment ofgeneralized fibrosis; as well as their uses and therapeutic applicationsin the treatment of fibrosis.

An advantage of greater importance in the treatment of the generalizedfibrosis, particularly of hepatic cirrhosis, is that the delivery oftherapeutic genes is carried out through tissue-specific recognition bythe way of administration employed.

Another advantage of the therapeutic uses of the invention, which isdirected initially to revert hepatic cirrhosis, is the treatment ofgeneralized fibrosis in other target organs susceptible to suffer fromit, including, without limitation, the treatment of fibrosis in lung,heart, skin, kidney, among others, in mammalian animals, including humanbeings.

Another object is the design of a technology to send genes efficientlyto livers of animals affected by cirrhosis that resemble two types ofcirrhosis that usually affect human beings (Alcoholic cirrhosis andPrimary Biliary Cirrhosis).

Another advantage resulting from the fibrosis treatment is thatrecombinant adenovirus does not induce lethal toxicity in none of theinjected animals with the vectors.

Another objective of the invention allows us to discriminate themodification of the staining reaction with X-Gal between the endogenoustissue -galactosidase activity and the bacterial -galactosidase inducedby the infectious action of the adenoviral vector. The use of the greenfluorescent protein permits us to verify the in vivo transduction ofdifferent organs in rats to verify if the vector administration wasappropriate, if the expression remains, and besides not killing theanimals it is possible to conduct follow up observation after surgery.

Finally, all this evidence let us suggest that our system comprises anefficient vehicle to deliver therapeutic genes such as humanmetalloproteases MMP-8 active and latent; MMP-1, MMP-2, MMP-9 andMMP-13; collagenase which degrade the deposited collagen excess and/orgenes which encode for promoters of hepatic regeneration such as humanuPA (urokinase Plasminogen Activator, modified and wild type),Hepatocite Grow Factor (HGF); the truncated receptor for TGF-β type IIand Smad 7 to livers of cirrhotic rats, with the purpose to re-establishnormal liver functions or normal functions of other organs affected bythe same pathology.

Thus, in the present invention a process of preparation is given,through which adenoviral recombinant vectors, pharmaceutical compoundsand therapeutic uses for the fibrosis treatment, especially for thetreatment of hepatic cirrhosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Other particularities and advantages of this invention will be evidentin the following detailed description of the preferred objects andembodiments, from the enclosed claims and from the drawings or shapesattached, in which:

FIG. 1 shows the cellular physiopathology of hepatic cirrhosis;

FIG. 2 shows the proof of concept on how gene therapy works by revertingthe cirrhosis process;

FIG. 3 is the schematic representation, which shows the cloning andproduction of the adenoviral vector Ad5-gal;

FIG. 4 shows the schematic development of the AdEasy system to generaterecombinant adenoviruses, specifically the pAdGFP-MMP-8;

FIG. 5 shows the analysis of the expression of -galactosidase incultured cells.

FIG. 6 shows the expression determination of green fluorescent protein(GFP) expression in cultured cells;

FIG. 7 shows the expression of -galactosidase in different organs afterthe infusion with Ad5 gal through the iliac vein.

FIG. 8 shows the analysis of the tropism of the vector Ad5-gal todifferent organs of cirrhotic experiment animals by chronic intoxicationwith CCl₄, demonstrating that the main target organ is the liver;

FIG. 9 shows the analysis of the tropism of vector Ad5-gal to differentorgans of cirrhotic experiment animals. Cirrhosis was induced by bileduct ligation and it was demonstrated that the main target organ is theliver.

FIG. 10 shows histological sections of representative images of the invivo efficiency transduction assays of the vector Ad5-gal in cirrhoticrats with chronic administration of CCl₄;

FIG. 11 shows histological sections of representative images of the invivo efficiency transduction assays of the vector Ad5-gal in cirrhoticrats by common bile duct ligation;

FIG. 12 shows the in vivo determination of the expression of the greenfluorescent protein;

FIG. 13 shows the cloning strategy of the latent MMP-8 and active MMP-8;

FIG. 14 shows the mechanisms of complex formation with DNA of MMP-8s forin vitro transfection essays in cells of hepatic origin (HepG2);

FIG. 15 shows the verification through electrophoresis in agarose gelsof the success of cloning of MMP-8 cDNAs in the appropriate plasmids;

FIG. 16 shows the transfection efficiency in HepG2 cells (Cells ofhepatic origin) with the plasmids of -galactosidase and cDNA-MMP-8;

FIG. 17 shows the analysis by polymerase chain reaction associated toreverse transcriptase (RT-PCR) of MMP-8 messenger RNAs;

FIG. 18 shows analysis of the collagenolytic activity in the proteinsecreted to the culture medium by HepG2 cells after transfection withcDNAs for latent MMP-8 and active MMP-8;

FIG. 19 shows the hormonal regulation of the MMP-8 gene expression underthe transcriptional control of the regulable promoter PEPCK and,

FIG. 20 shows the dose-response assay of the different doses used todetermine the best response of “in vivo” hepatic transduction with the-galactosidase reporter gene.

DETAILED DESCRIPTION OF THE INVENTION

There are many reports showing that through systemic administration ofrecombinant adenoviral vectors (AdR) into healthy experiment animals, aspecific homing and highly preferential tropism of these vectors intothe liver is observed. Up to now, it was not known whether the AdR wereable to transduce cirrhotic rat livers. As previously mentioned, hepaticcirrhosis is characterized by an increase of fibrosis in the entireliver parenchyma, mainly around the central and portal veins, creating abarrier which hampers the exchange of macromolecules between thesinusoid and the hepatocytes (Antoni P P, Hishack K G, Nayak N C,Poulsen H E, Scheuer P J, Sobin L H. The morphology of cirrhosis:Definition, nomenclature and classification. Bulletin of the WorldHealth Organization. 1977; 55:521-540; and Scott L. Friedman: Thecellular basis of hepatic fibrosis: Mechanisms and treatment strategies,The New England Journal of Medicine, 1993, Vol. 328, No. 25:1828-1835),and this protocol was designed to verify if even in presence of thisbarrier, the exogenous genes could be systemically delivered to thecirrhotic liver.

Therefore, our hypothesis is that AdRs containing LacZ and GFP (greenfluorescent protein) reporter genes are capable of transducing livers ofcirrhotic rats even if the lobular architecture of the liver isdistorted.

Thus, we could sent to these livers therapeutic genes such as humanmetalloproteases or collagenases human MMP-8 active and latent, MMP-1,MMP-2, MMP-9 and MMP-3; human Urokinase Plasminogen Activator (uPA wildtype and/or modified); the truncated receptor for TGF-β type II and Smad7, which degrade the excess of collagenic proteins deposited and/orprevent the exacerbated synthesis of collagenic proteins, as it is shownin FIGS. 2 and 18; and/or genes which encode for proteins stimulatinghepatic regeneration such as uPA, in order to re-establish the normalfunctioning of the liver, as is shown in FIG. 2.

The current invention initiates a research line to carry out genetherapy as an alternative for the treatment of chronic degenerativedisease, specifically of hepatic cirrhosis in human beings, through theestablishment of an efficient vehicle to send genes to the liver whichwill produce therapeutic proteins to help re-establish the normalfunctions of the liver, see FIG. 2. FIG. 2 shows how sending efficientlya therapeutic gene to the liver, in this case, a collagenase(metalloproteases of matrix, MMPs), it is possible to promotedegradation of collagen through the over-expression of thesemetalloproteases.

In FIG. 3, the strategy for the cloning and production of an adenoviralvector is shown. The plasmid pDeltaE1sp1B contains adenovirus Ad5sequences, in which the bacterial gene Lac-z was inserted. This plasmidwas recombined with the pBHG10 to obtain complete viral particles afterco-transfection in the cell line 293. The vector pAdGFP was obtained asfollows: the MMP-8 gene (coming from the plasmid PEPCK-MMP-8) was clonedin the vehicle vector, pAdTrack-CMV, the resultant plasmid is linearizedwith the restriction endonuclease Pme I, and is then transformed in E.coli (BJ5183) with the plasmid pAdEASY-1. The recombinant colonies wereselected through kanamicine resistance, and the recombination isconfirmed by restriction analysis with endonucleases. Finally, therecombinant plasmid linearized is transfected in the packaging cell line(293 cells), the recombinant adenoviruses are obtained within 7 to 12days as illustrated in FIGS. 3 and 4 (Tong Chuan H., Shibin Z., Luis T.Jian Y, Kenneth W. and Volgestein Bert: A simplified system forgenerating recombinant adenoviruses. Prod. Natl. Acad. Sci. USA Vol. 95:2509-2514, March 1998). To evaluate the grade of transduction in vitroliver HepG2 cell line and peritoneal macrophages isolated from mousewere used. In FIG. 5 the expression of -galactosidase in cultured cellsis shown. A), B) and C) correspond to HepG2 cells (32O×); D), E) and F),are mouse peritoneal macrophages (100×). In C) and F) the transducedcells are shown with 1×10⁸ viral particles/ml from the Ad5-Gal vector.Three techniques were conducted to compare the degree of incorporationof the reporter gene Lac-Z which was administered to each culture dishin the form of plasmidic DNA PGK-Gal, through precipitation with Ca⁺⁺phosphate (Chen C, and Okayama H. Calcium Phosphate mediated genetransfer, a highly efficient system to establish transforming cells withplasmidic DNA. Biotechniques 1988, 6:632-638), DNAcomplexes-polylysine-Lactose (Martinez-Fong D., Mullersman J E, PurchioA F, Armendariz-Borunda J., and Martinez-Hernandez A., Non enzymaticglycosylation of poly-L-lysine: A new tool for targeted gene delivery.Hepatology, Vol. 20, No. 6: 1602-1608), with the vectors Ad5-gal andpAdGFP-MMP8. The visualization of the activity of Gal was verified withthe reactive Xgal and the GFP in a microscope-stereoscope offluorescence. For the in vivo assay, gal staining was standardized usingdifferent pHs of the suspension with the reactive Xgal (Weiss D J,Ligitt D., and Clark J G. In situ photochemical detection ofgalactosidase activity in lung: assessment of Xgal reagent indistinguished Lac-Z gene expression and endogenous galactosidaseactivity. Human being therapy, Sep. 1, 1997, 8:1545-1554).

The models of experimental hepatic cirrhosis used are: a) Chronicintoxication caused by carbon tetrachloride (CCl₄), in which hepaticcirrhosis is established starting from the 8^(th) week of peritonealadministration (Mion F, Geloen A, Agosto E. and Minaire Y. Carbontetrachloride induced cirrhosis in rats: influence of the acute effectsof the toxin on glucose metabolism. Hepatology 1996, Vol. 23, No.2:582-587); and B), ligation of the bile duct (LCB) in which cirrhosisis observed after the fourth week of surgery (Lee S, Giraud C., DraillonA., HADengue A., and Lebec D., Hemodynamic characterization of chronicbile duct ligated rats; effect of pentobarbital sodium. AM Journalfisiol. 1986; 251:176-180; Nakano S., Harakane J. and Hashimoto H.,Alteration in peribiliary ducts microcirculation in rats after commonbile duct ligation. Hepatology, 1995, Vol. 21, No. 5: 1380-1995; DumasWalla R., Belcowitz D., and H. Eubi J E. Adaptive response of theEnterohepatic circulation of bile acid to extra hepatic. CholestiasisHepatology 1996, Vol. 23, No. 3: 623-629 and Poo J. L., Stanes A.,Pedraza-Chaverri J., Cruz C., Pérez C., Huberman A. and Uribe M:Cronologia de la Hipertensión Portal, Disminución de la Excreción desodio y activación del sistema renina-angiotensina en cirrosis biliarexperimental. Rev., Invest Clin, 49:15-23, 1997).

Ad5 gal was administered at the same time and from the same lot tocontrol rats without cirrhosis. Rats with 5 and 8 weeks of CCl₄intoxication and rats with 2 and 4 weeks of bile duct ligation (BDL)were sacrificed 72 hrs after administration of recombinant adenovirusfor the histological analysis and determination of the expression of thegalactosidase protein (gal) encoded by the AdR. For this purpose liver,spleen, heart, lungs, kidneys and brain were extracted, tissue sectionswere cut in cube shapes of 5 to 6 mm., which were absorbed in freezemedium Tissue-Tek O.C.T., the tissues were frozen at −30° C. and theywere cut with a cryostat to obtain 8 μm sections These sections wereplaced on silanized glass slides and fixed with formaline, pH 8.5,during 15-30 minutes and were exposed to Xgal for 16-18 hours, beingcounterstained with Neutral Red stain. (Weiss D J. Ligitt D. and Clark JG. In situ Hiti Chemical Detection of galactosidase activity in lung:assessment of Xgal reagent in distinguishing 1AC-Z Gene expression andendogenous galactosidase activity. Human Gene Therapy, Sep. 1, 1997,8:1545-1554). The percentage of positive cells was determined bymorphometric analysis in multiple fields of the same size andcalculating the average. Besides, liver sections of cirrhotic rats wereobtained and tissues absorbed in paraffin were cut and stained withSirius red which specifically stains collagenic proteins(Armendariz-Borunda J., and Rojkind M., A simple quantitative method forcollagen typing in tissue samples: Its application to Human liver withschistosomiasis. Collagen Rel. Res 1984, Vol. 4, 35-47). Through thistechnique we can verify clearly the degree of fibrosis and the increaseof bile ducts in the hepatic parenchyma. To verify the in vivotransduction of cells with GFP, we used healthy Wistar rats thatreceived pAdGFP-MMP-8 vector. 72 hours later, a laparotomy was performedand the exposed organs were visualised in the microscope offluorescence, closing the wound afterwards to keep the animal alive.

The previous results that are presented here regarding the study of thephysiopathology of experimental hepatic cirrhosis are summarized in FIG.2. Said figure shows the role of pro-inflammatory and pro-fibrogeniccytokines produced in vivo by Kupffer cells which, in turn, activate thehepatic stellate cells (HSC) to have them produce excess collagensdeposited in the subendothelial space, obstructing the exchange betweenhepatocytes and sinusoids (Armendariz-Borunda J., Katayama K., and SeyerJ. M.: Transcriptional mechanisms of type I collagen gene expression aredifferentially regulated by IL-1beta, TNFalfa and TGF into cells. J.Biol. Chem. 267:14316-14321, 1992; Armendariz-Borunda J., Katai H.,Jones C. M. Seyer J. M. Kang A. H. and Raghow R.: Transforming growthfactor beta is transiently enhanced at a critical stage during liverregeneration following CCL4 treatment. Laboratory Investigation.69:283294, 1993 and Armendariz-Borunda J., Roy N., Simjewish C., RaghowR. Seyer J. M. and Kang A. H.; activation of Ito cells involvesregulation of API collagen Gene Expression. Biochemical Journal304:817-824, 1994). The degree of incorporation of Lac-z gene incultured cells showed visible differences between techniques ofCalcium-Phosphate, DNA-polylysine-lactose complexes and with therecombinant adenoviral vector in HepG2 and PMM (Peritoneal mousemacrophages). The degree of transduction with adenovirus reaches 100%and with the other two techniques about 1% as shown in FIG. 5. FIG. 6shows the expression of green fluorescent protein (GFP) in culturedcells. A). Peritoneal mouse Macrophage transduced with the adenoviralvector pAdGFP-MMP8, 72 hours after its administration (50×), B). HepG2cells transduced with the adenoviral vector pAdGFP-MMP8, 72 hours afterits administration (50×) and C). HepG2 cells without the adenoviralvector. All the pictures were taken in a microscope stereoscope offluorescence. It is necessary to point out that in the development toidentify galactosidase activity, the cells must be fixed and they die.In the GFP assay, the cells are still intact and alive.

FIG. 7 shows the expression of gal in different organs after infusionwith Ad5 gal by iliac vein. Fixation, washing and Xgal solutions usingdifferent pHs were used to discriminate among the endogenous expressionand the bacterial exogenous galactosidase. In figure A, a pH 7.0 wasused and in Figure B the pH was 8.5. This is the summary of the resultsof the assays of the different experimental conditions and it can beappreciated that the tissue exposition to Xgal solution with a pH 8.5allowed us to eliminate the expression of endogenous galactosidase. Weobtained frozen tissue sections from different organs: liver, kidney,lung, heart, brain and spleen from normal rats and intoxicated with CCl₄for five and eight weeks. As represented in FIG. 8, the graphics showclearly that the main target organ is the liver, both in healthy rats aswell as in rats with chronic administration of CCl₄. A) 5 weeks of CCl₄administration and B) 8 weeks of CCl₄ administration. Spleen and lungpresent a degree of transduction below 1%, and thus this is not evidentfrom the graphs. Rats received doses of 3×10¹¹ viral particles/ml ofAd5gal vector. The healthy control rats presented a total of 70% ofhepatocytes transduced, while spleen and lung showed less than 1%transduction. In the other organs no transduction was found. Tissuesections were obtained from healthy rats as described before andcompared with tissues from rats with 2 and 4 weeks of BDL. FIG. 9clearly shows how the main target organ is the liver, both in healthyrats as well as in BDL rats. A) 2 weeks of LCB and B) 4 weeks of BDL.The spleen and the lung present a transduction grade lower than 1%, andthus it is hardly noticeable in graphs. With a dose of 3×10¹¹ viralparticles/ml of the AD5gal vector, BDL rats present a total of 10%transduced hepatocytes. Besides liver, spleen and lung presented lessthan 1% transduction. The other organs showed no transduction. In FIG.10, histological results are shown with the hepatic cirrhosis modelinduced by the chronic administration of CCl₄, where A) represents aliver section of a normal rat, 72 hours after the administration of Ad5gal, by iliac vein (one representative cut of the experiments of a totalof 5 rats). More than 70% of the hepatocytes are positive to theexpression of gal (200×); D) The same liver as in Figure A, but stainedwith Sirius Red to observe collagen synthesis and deposition (200×); B)liver with 5 weeks of chronic intoxication with CCl₄. About 30-40% ofthe hepatocytes were successfully transduced; E). The same livers as inB, but stained with Sirius Red, the increase in the amount of collagenis notable and the liver architecture begins to distort (200×); C) ratliver after 8 weeks of chronic intoxication with CCl₄ to causecirrhosis, again more than 40% of liver cells were positive for βgalexpression and F) the same livers as in C, but stained with Sirius Red.Large deposits of collagen formed between the central and portal veins(200×) are characteristic. In FIG. 11, results obtained in the model ofbiliar duct ligation (BDL) induced cirrhosis are shown. A) shows a liversection of a normal rat 72 hours after the administration of Ad5 gal, byiliac vein (one representative cut of the experiments of a total of 5rats). More than 70% of the hepatocytes are positive to the expressionof gal (200×); D) the same liver as in Figure A, but stained with SiriusRed to observe collagen (200×); B) rat liver after 2 weeks of BDL. β-galessay was conducted 72 hours after Ad5βGal administration, via iliacvein. About 10% of the hepatocytes were successfully transduced with thereporter gen; E) the same livers as in B, but stained with Sirius Red.Liver architecture begins to distort due to colestasis-induced fibrosisas well as to the important increase of biliar ducts (200×); C) ratliver after 4 weeks of BDL to cause cirrhosis. β-gal essay was conducted72 hours after the administration of Ad5βGal, via iliac vein. Again, 10%of hepatocytes were successfully transduced and F) the same livers as inC, but stained with Sirius Red. Observe the large deposit of collagenproteins formed as well as the proliferation of biliar ducts (200×).FIG. 12 shows a laparotomy of a healthy Wistar rat that receivedpAdGFP-MMP-8 vector. The expression of the GFP is clearly seen in theliver and in insignificant amounts in the spleen. A very important factis that the injection of adenoviral vectors did not induce lethaltoxicity in experiment animals, both healthy and controls.

The preferred way to apply the present invention is through endovenousadministration of the recombinant adenoviral vectors of this inventionor the pharmaceutical compound which contains them, in whichtherapeutically effective amount is administered with an unitary doseregimen convenient to an individual with fibrosis. This regimen can beadjusted according to the affliction degree. Generally, unitary doses ofabout 10⁷ to 10¹⁴ viral particles for individual are employed. Thepreparation of a pharmaceutical compound including the adenoviralrecombinant vectors of this invention can be conducted through theemployment of standard techniques very well known by the persons skilledin the art, in combination with any of the pharmaceutically acceptablecarriers described in the state of the art, including withoutlimitation, starch, glucose, lactose, saccharose, gel, malt, rice, wheatflour, chalk, silica-gel, magnesium stearate, sodium stearate, powder ofglyceril monostearate, NaCl, glycerol, propylene glycol, water, ethanol,and similar. These compounds can take the pharmaceutical form ofsolutions, suspensions, pills, tablets, capsules, powders and slowrelease formula, and similar.

The above description and the following examples have the purpose toillustrate particular embodiments of the invention and they should notbe considered as limitations of the scope of this patent.

EXAMPLES Example 1 Methodology to Demonstrate the Activity ofMetalloprotease or Collagenase (MMP-8) and How to Regulate its Function

a) Cell culture. HepG2 cells is a cell line of parenchymal originderived from a human hepatoma, and were cultured in 60 mm culture dish,37° C. in a wet atmosphere, with 95% air and CO₂ 5% atmosphere inEagle's medium modified by Dulbecco (DMEM), supplemented with 10% fetalbovine serum, 2 mM L-Glutamax and antibiotics (100 U/ml penicillin and100 g./ml. streptomycin).

b) Vectors of Expression of Latent and Active MMP-8 Genes

Two plasmids were used with 2 kinds of MMP-8 genes to transfect thehepatic cells: The plasmid pcDNA-MMP-8 which contains the cDNA whichencodes for latent MMP-8 (pro-MMP-8) together with the strong viralpromoter of cytomegalovirus (CMV) (ATCC Deposit No. PTA 10532); and theplasmid pcDNA3MMP-8 containing the cDNA which encodes for the activeMMP-8, together with the CMV promoter. This last one was created throughsubclonation using pcDNA3 and PETIIa-HNC plasmids, cutting with therestriction enzymes BamHI and XbaI and inserting the PCR product codingfor the MMP-8 catalytic domain (which lacks the propeptide andcarboxi-terminal fragments), as shown in FIG. 13, the delivery of latentand active MMP-8 genes. Two types of plasmids with the MMP-8 gene wereused to be delivered to hepatic cells in culture: 1) PcDNA3-MMP-8,plasmid with the strong viral promoter of the cytomegalovirus (CMV) andthe cDNA which encodes for the collagenase in its active form.

As a reporter gene pSV₂-gal plasmid was used. Said plasmid has the genewhich encodes the enzyme -galactosidase inserted adjacent to the SV40virus promoter.

c) Plasmid Transformation, Amplification and Purification

To obtain a large enough quantity of each one of the plasmids to be usedin the various assays, each plasmid was introduced to E. coli DH5™,(this process is known as transformation), according to the instructionsof the supplier. (Life Technologies, Gaithersburg, Md.): in a reactiontube 50 l of the competent strain DH5 were used and 2 l of plasmids(1-10 ng of DNA) were added. After mixing, it was incubated on iceduring 30 minutes, a thermal shock (37° C. for 20 seconds) was appliedand it was immediately chilled on ice for 2 minutes. At the end of thisperiod of time, 0.95 ml of the bacterial culture medium Luria Base (LB)was added and it was stirred at 225 rpm during one hour to 37° C. toallow plasmid expression. After the expression, 50 l of the reaction mixwere taken and seeded onto an Agar plate with 100 g/ml of ampiciline andit was incubated to 37° C. overnight. The colonies that grow after thisperiod are those which contain the plasmid of interest, because of theresistance against the antibiotic.

To amplify the plasmid, two colonies were taken from the Agar plate andgrown in a liter of LB medium containing 100 g./ml of ampiciline during24 hours at 37° C., with constant stirring at 225 rpm. Once the opticdensity of the culture was 0.6, it is centrifuged to 6,000 rpm for 20minutes to recollect the bacterial pellet. From this bacterial pellet,plasmidic DNA was separated from the genomic DNA of the bacteria using akit of plasmids purification (Monster-prep, BIO101, Vista, Calif.),which is based on the alkaline lysis of the bacterial wall, theliberation of the plasmid in its interior and the separation of this DNAthrough a particular resin. The quantification of the plasmidic DNA wasperformed measuring spectrophotometrically the resultant absorbance atλ=260 nm.

d) Transfection of Cultured Cells

One of the most commonly used methods to introduce genes to eucaryoticcells, is DNA transfection with calcium phosphate, in which theexogenous DNA is precipitated as a fine complex on the cell surface, tobe later incorporated by the cell and transiently integrated in thechromosomal DNA. To deliver the DNA with more selectivity to the hepaticcells, DNA is used in the form of complex with polylysine-lactose,because of hepatic cells have a specific receptor for Galactose in theircell membrane. For this, HepG2 cells were cultured at 70-80% confluenceand then transfected with plasmids pcDNA-MMP-8, pcDNA₃-MMP-8 andpSV₂-galactosidase. Transfection was carried out by DNA precipitationwith calcium phosphate (Graham, and Van derEb, 1973; Chen and Okayama1988) and by complex formation with polylysine-lactose (Martinez-Fong etal, 1994). Briefly, cultured cells were added with the newly formedprecipitate, product of the addition to plasmidic DNA of a solution ofDNA with CaCl₂ 2M, in buffer solution HEPES pH 7.12 in case of thetransfection with calcium phosphate or DNA complex withpolylysine-lactose is added. Cells are incubated from 4-16 hours toallow the precipitate to appear to the cell surface, and later the DNAcan be endocyted and introduced transiently to the nucleus. At the endof this time, the culture medium is replaced for a fresh one, see FIG.14, where HepG2 cells are cultured with DMEM medium with 10% bovinefetal serum. When 60-80% confluency is reached, 10 mg of plasmid withMMP-8 gen is added in its latent form, as well as in the active ormature form. At the same time, the prokaryotic gene of galactosidase(-gal), is added to monitor the transfection and expression efficiency.MMP-8 gene was sent in different forms: naked, in complex with CaPO₄ orin complex with polylysine-lactose.

e) Formation of Complexes Polylysine-Lactose and DNA: Polylysine-Lactose(DNA:PL)

The polylysine-lactose complex is formed when 14.8 mg of poly-L-Lysine(0.1 N) react with 200 l of -lactose 0.5 N (lactose-polylysine ratio:1.0 N). Then, 20 mg of reducing agent sodium cyanoborohydride 3 M isadded and it is incubated at 37° C. for 48 hours with constant stirringat 225 rpm. Then, the reaction goes through a desalting column (BioRad10-DG) previously conditioned with phosphate buffer (PBS pH 7.2), whichis eluted with the same buffer. Carbohydrate content is determined tothe eluted fractions by the method of DuBois (1956) to analyze thedegree of lactosylation of the complex and the contents of polylysineaccording the method of Shen et. al. (1984), which is considered as abase to evaluate the final concentration of the PL complex. The fractionwith a major concentration of PL is used for its further reaction withthe DNA of the plasmid containing the gene of interest, as is shown inFIGS. 14 and 16.

To evaluate the optimal molar ratio of DNA:PL to be used in transfectionassays, the DNA was made to react with several concentrations of PL. Atthe end of one hour of incubation, samples were applied to a 1%retardation agarose gel and submitted to electrophoresis of DNA (60millivoltios, 1.5 h), in which the DNA:PL complex with the largest PLcontents runs a shorter distance than the one run by the free plasmid(0% retardation). The DNA:PL ratio which causes 80 to 90% of retardationof migration in the agarose gel was used as shown in FIG. 16 to obtainan efficient expression of exogenous genes of -galactosidase andpcDNA-MMP-8 delivered to HepG2 cells in complexes with CaPO₄ andpolylysine-lactose.

f). Assays of Transient Expression Using the Reporter gen System of-Galactosidase (gal)

This system determines the activity of the -galactosidase enzyme as ameasure of the level of expression of the transfected gene of interestalong with Lac Z gene which encodes for this enzyme. The galactosidaseis a bacterial enzyme which catalyzes the conversion of the uncoloredsubstrate X-gal to a product of blue coloration. Because of this, the-galactosidase activity observed in eucaryotic cells subjected totransfection will indicate the successful incorporation of the gene ofinterest associated to the bacterial gene.

The assay of -gal for the stain of cells in culture dish consists in thefixation of cells at 4° C. during 5 minutes with 2% p-formaldehyde, thesubsequent wash with PBS (3×) and the addition of one ml of a stainsolution in PBS containing 20 mM potassium ferricianide, 20 mM potassiumferrocianide and 2 mM Magnesium Chloride followed by the addition of thesubstrate Xgal in a final concentration of 0.5 mg/ml. After incubationat 4° C. overnight (18 hours) blue cells are identified under themicroscope (Ausubel, 1995).

g) RNA Extraction

48 hours after transfection, cells are recollected to extract RNA by theMethod of Chomczynski and Sacchi (1987) using the reactive of Trizol™,as described hereinafter: to each one of the cell dishes one ml of PBSsolution was added and cells were recollected by scraping them from thedish and transferred to an Eppendorf tube. It was then centrifuged at1000 rpm for one minute and the cell pellet was treated with 500 l ofTrizol, homogenized and incubated for 5 minutes at 4° C. One hundred μlof chloroform were added, and incubation was conducted during 5 minutesat 4° C. After this, it was centrifuged at 12,000 g for 15 minutes at 4°C. and the aqueous upper phase was transferred to a clean tube in whichan equal volume of isopropanol is added and incubated at −70° C. during15 minutes to precipitate the extracted RNA. Then, it is centrifuged at12,000 g during 15 minutes at 4° C., the supernatant is eliminatedthrough decantation and drying the tube with clean and sterile paper.Then, 500 l of 75% ethanol were added and it was centrifuged at 12,000 gduring 10 minutes to 40° C. Finally, the RNA pellet was resuspended with20 to 50 l of deionized water treated with diethylpirocarbonate (DEPC)and RNA concentration was quantified by spectrophotometry at λ=260 nm.

h) Analysis of Expression of MMP-8 Gene by the Polymerase Chain Reaction(PCR) Associated to the Reaction of Reverse Transcriptase (RT-PCR).

To determine the degree of expression of the exogenous gene of MMP-8incorporated to the cell, complementary DNA was obtained (cDNA) startingfrom RNA previously extracted and then amplifying the expression signalby the Polymerase Chain Reaction.

To obtain the cDNA, the following procedure was used: 2 g of total RNAwere taken to a volume of 8 l with deionized, sterilized water andincubated at −70° C. for 10 minutes. Then, the sample was stirred iniced water during 5 minutes and still in the ice, the following reagentswere added: 4 l of 5× buffer for the RT enzyme, 4 l dNTP's mix 2.5 mM, 1l random primers (1 g/l), 1 l inhibitor of RNAase (one U/l) and finally2 l of the Reverse Transcriptase enzyme (200 U/l). The reaction mix wasincubated at room temperature for 10 minutes and then at 37° C. for onehour. At the end of this time, it was placed immediately in atemperature of 95° C. for 10 minutes, and then it was placed on icedwater during 5 minutes with constant stirring and it was stored at −70°C. until its further use.

To analyze the specific expression of MMP-8 gen, a PCR reaction was setup using the primers or oligonucleotides specific for this geneaccording to the experimental conditions described hereinafter: in areaction tube containing 2 l of cDNA 5 l of 2.5 mM MgCL₂, 5 l 5× bufferfor the polimerase enzyme, from leukemia murine virus moloney (MMLV), 1of 2.5 mM dTNPs, 5 l of the sense primer 3 μM, 5 l of the antisenseprimer 3 μM, 1 l of the polymerase enzyme (U/l) and it is taken to afinal volume of 50 l with deionized water (Innis et al. 1990). Theoligonucleotide sense primer specific for MMP-8 is5′-AGCTGTCAGAGGCTGGAGGTAGAAA-3′ (SEQ. ID 1), and the antisense primer is5′-CCTGAAAGCATAGTTGGGATACAT-3′(SEQ. ID 2) (Cole et al. 1996). After theaddition of these reagents, the mix was placed in a thermalcycler during30 cycles according to the following program: denaturation (94° C., 5min), annealing (60° C., 1 min) and extension (72° C., 1.5 min). Then,PCR products are submitted to electrophoresis (60 mV, 1.5 h) in a 1.5%agarose gel.

i) Assay of Collagenase Activity.

The analysis of enzymatic activity of collagenase was performed todetermine the functionality of the enzyme produced, because this proteincould be found enzymatically inactive, even when RNA expression waspositive. Cells are cultured in serum-free medium for 24 hours, culturemedium is recollected and activity of collagenase secreted by the cellsis determined by a modified method of Hasty et al. (1986) to identifyproducts of degradation of specific collagen substrate through 8%polyacrylamide gel electrophoresis.

Briefly: cell supernatants containing 1-1.5 gr of protein were incubatedat 27° C. during 18 hours with 5 g of native collagen type I and 60 l ofthe incubation buffer: 50 mM Tris-HCl, 5 mM CaCl₂, 0.02% NaN₃, 50 mMarginine, 1% Triton X-100 and in absence or presence of 1 mM APMA, pH7.6. Finally, 30 l of product of reaction were mixed with 30 l of samplebuffer for proteins and electrophoresis in SDS-polyacrylamide gels(7.5%) was run to identify the degradation products 1^(A) and 2^(A) ofcollagen type 1.

Example 2 Results to Demonstrate the Activity of Metalloprotease orCollagenase (MMP-8) and Therefore to Regulate its Function

Subcloning permitted to incorporate MMP-8 cDNA encoding for the fullyfunctional enzyme was subcloned in a vector appropriate to our needs.Thus, FIG. 15 shows an electrophoresis of the DNA fragments released bycutting MMP-8 plasmids with restriction enzymes. Lane A). Marker of bpof 1 Kb DNA ladder (Gibco BRL); B). Perfect DNA marker (Novagen,Inc.); 1) pcDNA-MMP-8 cutting with BamHI and XbaI; 2) pcDNA3-MMP-8cutting with BamHI and XbaI; C) φX174 marker (Gibco BRL); λ Hind IIIMarker (Gibco BRL), in which the latent MMP-8 cDNA (lane 1) and themature MMP (lane 2); were successfully subcloned in the expressionvectors pcDNA and pcDNA3. The released inserts are observed aftertreatment with restriction enzymes BamHI and XbaI. The bands stainedwith ethidium bromide correspond to each of cDNA (between 506 and 560base pairs) for mature and latent MMP-8 cDNA, respectively. To evaluatethe efficiency of incorporation of the cDNA for MMP-8 delivered to HepG2cells in form of complex with CaPO₄ and with polylysine-lactose, theco-transfection of this plasmid was realized along with the reportergene of -galactosidase. In this way, cells observed in the microscopewith blue staining, indicate indirectly that they have also incorporatedto the plasmid of interest. FIG. 16 shows the expression of-galactosidase in HepG2 cells, co-transfected with free plasmid, in formof complex with CaPO₄, or in its form of complex withpolylysine-lactose. This figure shows that the DNA binding withpolylysine-lactose was accomplished because the higher the polylysineconcentration, the clearer the retardation of -gal plasmid. The ratioselected to transfect the cells was the one that delayed 80% of plasmidmigration.

Once demonstrated that the cells in culture are capable of incorporateand express genes that have been transfected, it was necessary tocorroborate that such genes were transcribed by the machinery of hostcells by means of RT-PCR assays. FIG. 17 shows an analysis by RT-PCR ofmessenger RNA for MMP-8 and MMP-13. (This plasmid was used as a furtherpositive control of transfection); in which a DNA electrophoresis of PCRamplified products, of the cDNA for MMP-8 delivered as a complex withCaPO₄ and polylysine-lactose, has been transcribed for both cases intransfected HepG2 cells. It is observed that product signal of PCR ofMMP-8 (359 base pairs), was more intense when plasmid was delivered as acomplex with polylysine-lactose.

To demonstrate that MMP-8 transcripts expressed by HepG2 cells wastranslated into a functional protein, the assay for enzymatic activitywas conducted, using collagen type I as substrate. FIG. 18 shows theenzymatic activity of type I collagen degradation of the proteinsecreted in the culture medium, which was observed in the transfectedcells with the gene of latent MMP-8. With previous activation with themercurial agent APMA (lane 7) and with the gene of active MMP-8complexed with CaPO₄ (lane 9) and with polylysine-lactose (lane 10), andits specific inhibition with EDTA 2 mM. Negative controls: type Icollagen without addition of supernatants of cells (lane 1) and withaddition of Trypsin (lane 3), collagen with supernatants of cellswithout transfection (lane 2). Positive controls: type I collagen withsupernatant of human leukocytes (lane 3), type I collagen with additionof 0.015% bacterial collagenase (lane 4); and degradation products ofnative type I collagen, separated in a 6% polyacrylamide gel, after itwas incubated with supernatant of transfected cells with latent andactive MMP-8 genes. It was observed how in both cases the collagenolyticactivity is clear in presence of APMA in the case of latent MMP-8, andits inhibition for EDTA for both latent and active MMP-8. This factshows that this proteolytic activity corresponds to a metalloprotease ofinterstitial matrix. The incubation of native type I collagen withtrypsin did not show degradation. So, this experiment clearly shows thatMMP-8 action was specific considering the intact nature of the collagenmolecule.

FIG. 19 shows evidence that activities of the enzymes that specificallydegrade collagen can be controlled (turned off and/or turned on) throughthe cloning of its respective cDNAs that are themselves under thetranscriptional control of promoters of regulable genes, such as thePEPCK (Phosphoenol-piruvate carboxykinase) gene. It is clear that boththe stimulation of cells in culture with Glucagon (lanes 5 and 6), andcyclic AMP (lanes 7 and 8), up-regulate their production of messengerRNA that codes for MMP-8. It is also clear that insulin lowers saidproduction (lanes 9 and 10).

The observations regarding the activity of endogenous -galactosidasesuggest that this activity is usually granular and weaker in color thanthe dark blue as a result of the activity of exogenous enzyme (ShimohamaS., Rosenbergh M B. Fagan A M, Wolff J A, Short M P, Bradfielf X O,Friedman T., and Gage F H: Genetically Modified Cells into the ratbrain: Characteristics of E. coli-Galactosidase as a reporter gene.Brain Res. 5:271-278, 1989). Many modifications have been described toincrease the specificity in the determination of exogenous Lac Z geneessay. Thus, according to previous information by Weiss, D J, Ligitt D.,and Clark J G. In situ histochemical detection of beta-galactosidaseactivity in lung. Assessment of Xgal reagent in distinguishing Lac Zgene Expression and endogenous -galactosidase activity. Human GeneTherapy, Sep. 1, 1997, 8:1545-1554; in the present invention a solutionof X-gal, with a pH 8.5 was used; in this way, the activity of exogenous-gal was demonstrated, minimizing the endogenous activity in vivo.

One of the indicators actually used for in vivo monitoring theefficiency and location of transduced cells with recombinantadenoviruses, is the detection of green fluorescent protein (GFP)expression. For this purpose, the gene which encodes for this protein issubcloned in adenoviral vectors, and then through the use of afluorescent microscope, the fluorescence given by GFP can be observeddirectly without sacrificing the experiment animal which received thevector (Rojas-Martinez, A, Wyde P R, Montgomery Calif., Chen S H, Woo SL C and Aguilar-Cordova E.: Distribution toxicity and lack ofreplication of an E1A-recombinant adenoviral vector after systemicdelivery in the cotton rat. Cancer Gene Ther. 1998, y TongChuan H.,Shibin Z., Luis T., Jian Y., Kenneth W., and Vogelstein Berth: Asimplified system for generating recombinant adenoviruses. Proc. Natl.Acad. Sci. USA Vol. 95:2509-2514, March 1998). A large body of data hasbeen obtained that shows that, after the i.v. administration ofadenoviruses in healthy animals, the main target cells were hepatocytes.This has been observed in mice, rabbits, dogs and primates (Zern A M.and Kresina T F, Hepatic drug delivery and gene Therapy. Hepatology1997, vol. 25, No. 2, 484-491), but not in cirrhotic rats. Probably, theinjection in portal vein could be more efficient to get to the targetcells in the liver, providing them a favorable innoculum of viralparticles to the entire liver before being diluted into the bloodstream.This route is efficient, but it has the disadvantage that it requires alaparotomy. On the other hand, peritoneal administration is a faster andsimpler infusion, but it does not promote hepatocyte transduction. Theresults of the present invention show that the injection of 3×10¹¹ viralparticles by iliac vein in normal Wistar rats of approximately 200 g.produces a very high level of expression (70% of transducedhepatocytes). Our results are consistent with a previous report in whichspecific delivery of reporter genes in primates by saphenous veinproduced almost the same level of transduction and expression of thetransgene in the liver, as compared with infusion through portal vein(Marie Jean T F D, Poeters V., Lieber A., Perkins J., and Kay M A.Methods for multiple portal vein infusion in mice: Quantitation ofadenovirus-mediated hepatic gene transfer. Biotechniques February 1996,20; 278-285 and Zhu G. Nicholson A G. Zheng X., Strom T B, and Sukhame VP. Adenovirus mediated -galactosidase gene delivery to the liver leadsto protein deposition in kidney glomeruli. Kidney international, 1997,Vol. 52, 992-999). Furthermore, the expression of the reporter gene inour animals with cirrhosis induced by chronic administration of CCl₄ wassurprisingly almost as high as the normal rats (40% of transducedhepatocytes). These results are very exciting because our cirrhoticanimals could hardly survive the surgical procedure required toadministrate the adenovirus by the portal vein. This is due to alteredfunctional hepatic tests, and elevated prothrombin time as well asimportant bleeding. Although rats with bile duct ligation showed asubstantial reduction in the number of transduced hepatocytes (5-10%),it is also important the number of hepatocytes, which eventually couldbe transduced with therapeutic genes, such as metalloproteases (MMP-8)and/or genes which encode for stimulating proteins for hepaticregeneration such as uPA (Urokinase Plasminogen Activator) and Smad 7.

Other embodiments will be evident for people skilled in the art based onthe present description. Said embodiments are included within the truescope and spirit of the invention.

The definitions of the symbols used in the figures corresponding to thepresent invention, are shown below:

FIG. 1: CEH = stellate hepatic cell. CES = Endothelial sinusoidal cell.CK = Kupffer Cell. ESET = Subendothelial space. HE = Hepatocytes. HIDC =Liver with chronic damage. HN = Normal liver SINU = Sinusoid FIG. 2:COLASA = Collagenase DCA = Degradation of collagen. TGE = Experimentalgenic therapy MMPs = Metalloproteases FIG. 3: CT293 = Co-transfection incells 293 PG CsCl = Purification with CsCl gradients FIG. 4: BD = Rightarm. BI = Left arm. CTBK = Co-transfection in bacteria and selection inKanamicine. CUL = Culture LI PacI = Linearize with Pac I. LI PmeI =Linearize with Pme I PV = Viral particles T293 = 293 Cell transfectionGENADR = Generation of recombinant adenovirus. FIG. 7: B = Spleen. CE =Brain CO = Heart % CT = Percent of transduced cells H = Liver P = Lung R= Kidney Sad -gal = Without the Ad -gal vector CAD -gal = With the Ad-gal vector X-GAL7 = Reactive X-gal, pH 7.0 X-GAL 8.5 = Reactive X-gal,pH 8.5 FIG. 8: B = Spleen. CCl45 = 5 weeks of intoxication with CCL4CCl48 = 8 weeks of intoxication with CCL4 CE = Brain C0 = Heart % CT = %of transduced cells H = Liver P = Lung R = Kidney PV = Viral particlesHN = Normal Liver FIG. 9: B = Spleen CE = Brain CO = Heart % CT = % oftransduced cells H = Liver LCB2S = 2 weeks of bile duct ligature LCB4S =4 weeks of bile duct ligature P = Lung R = Kidney PV = Viral particlesHN = Normal Liver FIG. 13: PROT = Protein APMA: MERCURIAL AMONOPHENYLACETATE FIG. 14: ACT -gal = - galactocidase activity CES = Cells EAC =Enzymatic Activity PL = Polylysine PROT = Protein RGAL = Galactoseresidues SNAD = Supernatant FIG. 16: ADND = Naked DNA. GELRADN-PL =Retardation gel for polylysine FIG. 18: CA = With APMA. CACE = With APMAand EDTA. CaPO4 = Phosphate. CE = With EDTA. COB = BacterialCollagenase. COL1 = Type I Collagen PL = Polylysine SA = Without APMASNL = Leucocyte Supernatant ST = No-transfected TRIP = Trypsin FIG. 20:% CT = % of transduced cells. PV = Viral particles.

1. A method of delivering matrix metalloproteinase 8 (MMP-8) to theliver of a subject in need of delivery thereof, the method comprisingdelivering a composition by an intravenous administrative route to thesubject, the composition comprising a pharmaceutically compatiblecarrier and unitary doses of viral particles of a recombinant adenoviralvector, wherein said unitary dose is from about 10⁷ to about 10¹⁴ viralparticles and the adenoviral vector is the vector contained in ATCCDeposit No. PTA-10532.